Macrophages are a primary cellular target of anthrax lethal toxin (LT), a bacterial secreted A-B toxin comprised of the proteins protective antigen (PA) and lethal factor (LF). PA and LF assemble on macrophage surfaces and are internalized by endocytosis. In a pH-dependent translocation process, PA delivers LF across endosomal membranes into macrophage cytosol where the protease activity of LF generates signals that lead to rapid death of macrophages, and possibly also to release of the inflammatory cytokines TNFalpha and IL-1beta. Experimental models of inflammation and bacterial infection have described a macrophage-specific, inflammatory cell death pathway, called pyroptosis that requires the protease caspase-1. Similarities between LF toxicity and pyroptosis lead to the hypothesis that cytosolic LF activates caspase-1 in macrophages, followed by cell death and release of the cytokines IL-1beta and IL-18. Addressing three specific aims will test this hypothesis: Aim 1. Define the roles of caspases in LF-mediated macrophage cytotoxicity and cytokine secretion. We will define the relationship between LF toxicity and caspase-1- dependent pyroptosis. Caspase activities will be measured in macrophages exposed to LF or ATP, or infected with S. typhimurium. LF-mediated cytotoxicity and cytokine release will be measured to determine the protective effects of specific caspase inhibitors and inhibitors of ATP-mediated cytotoxicity and cytokine secretion. Aim 2. Develop FRET-based probes of intracellular protease activities. An LF probe will be developed, consisting of cyan fluorescent protein (CFP) and citrine (a modified yellow fluorescent protein) linked by an LF protease substrate bridge. The intact molecule will exhibit fluorescence characteristic of FRET; and proteolytic cleavage of the bridge by LF will lead to loss of FRET. The LF probe will be purified for in vitro characterization, and will be expressed in macrophages for in situ characterization. It will then be used to define the timing of LF activity, relative to other physiological changes, in individual cells and populations of macrophages. To detect downstream signals generated by LF, analogous FRET -based probes of calcium and caspases-1, -3 or -8 will be developed, tested and employed. Aim 3. Define the temporal order of LF signals in sensitive and resistant macrophage. Using fluorescent probes for LF, calcium, and caspases, as well as biochemical assays for TNFalpha, IL- 1beta, IL-18, reactive oxygen intermediates and cytotoxicity, we will determine the chronology of events associated with LF signaling in macrophage cytoplasm. The temporal order of signaling will also be defined by timed addition of signal inhibitors. Essential pathways leading to cytotoxicity will be defined by comparing the various LT responses in macrophages from sensitive and resistant strains of mice. These studies will lie out the essential pathway of LT- dependent macrophage cytotoxicity and address important outstanding questions about macrophage cytokine responses. In doing so, they will guide the selection and design of effective, late-stage therapeutics.